WO2012077076A1

WO2012077076A1 - Treating or preventing sensitivity to milk allergens

Info

Publication number: WO2012077076A1
Application number: PCT/IB2011/055537
Authority: WO
Inventors: Patrick G. Holt
Original assignee: Holt Patrick G
Priority date: 2010-12-08
Filing date: 2011-12-08
Publication date: 2012-06-14

Abstract

The present invention relates to methods and compositions for promoting a beneficial immune response in a subject in need thereof. In particular the present invention relates to methods of decreasing the risk of a subject developing an adverse immune response to one or more milk allergens, such as one or more milk proteins.

Description

Treating or preventing sensitivity to milk allergens

FIELD OF THE INVENTION

BACKGROUND TO THE INVENTION

The ability of the immune system to respond appropriately to an immunological challenge is critically important to mammalian health and well-being. For example, the immune system plays a fundamental role in the body's response to pathogenic challenge, and the involvement of the immune system in the prevention of many diseases is well-recognised.

However, the immune system can also respond inappropriately or disadvantageously to challenge. Just as an appropriate immune response is recognised as a hallmark of immune health, an inappropriate immune response— for example, an allergic response to an allergen— is recognised as an indication that immune health is sub-optimal.

There have been suggestions that the global incidence of adverse immune responses, such as allergic responses to food allergens, is increasing. This may in part reflect an increase in the exposure of populations to new sources of allergens, for example, through increased intake of new or relatively new foodstuffs. Strategies to prevent or treat inappropriate immune responses to allergens, such as food allergens, have focussed on preventing exposure to the causative allergen, and have, to date, been largely unsuccessful, certainly in diminishing an individual's risk of an adverse reaction should exposure actually occur.

It is an object of the present invention to overcome or at least ameliorate some of the abovementioned disadvantages, to provide a method of establishing an effective and appropriate immune response useful in the prevention or treatment of sensitivity to one or more milk allergens such as one or more milk proteins, to provide methods for minimising the risk of developing sensitivity to one or more milk allergens in subjects in need thereof, or to at least provide the public with a useful choice.

Other objects of the invention may become apparent from the following description which is given by way of example only.

SUMMARY OF THE INVENTION

In one aspect the invention relates to a method of decreasing a subject's risk of developing sensitivity to one or more bovine milk allergens, the method comprising orally administering to the subject one or more bovine milk allergens, wherein the administration is of a composition comprising one or more bovine milk allergens wherein the one or more bovine milk allergens is present at a concentration at least 50% greater than the concentration (s) of the milk allergen(s) in whole bovine milk; and optionally wherein the administration is one or more of the following: a) of an amount sufficient to elicit tolerance to one or more bovine milk allergens; or b) of a frequency sufficient to elicit tolerance to one or more bovine milk allergens; or c) of a duration sufficient to elicit tolerance to one or more bovine milk allergens; or d) commences prior to any previous direct exposure of the subject to bovine milk or one or more bovine milk allergens (such that the administration of the one or more milk allergens represents the first direct exposure of the subject to the one or more milk allergens); or

e) commences prior to established sensitivity to one or more bovine milk allergens; or f) any combination of two or more of a) to e).

In one embodiment, the one or more bovine milk allergens is one or more components of bovine milk capable of increasing IgE production or levels in a subject exposed to same.

In one embodiment, the one or more bovine milk allergens is one or more components of bovine milk capable of increasing IgGl production or levels in a subject exposed to same.

In one embodiment, the one or more bovine milk allergens is one or more milk proteins, one or more milk protein fragments, including one or more allergenic peptides derived from a bovine milk protein.

In one embodiment, the one or more bovine milk proteins, milk protein fragments, or allergenic peptides is selected or derived from alpha_si-casein, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, or lactoferrin.

In a further aspect the present invention relates to a method of modulating in a subject in need thereof an immune response to one or more bovine milk allergens, the method comprising administering to a subject not previously sensitised to one or more bovine milk allergens a composition comprising one or more bovine milk allergens wherein the one or more bovine milk allergens is present at a concentration at least 50% greater than the concentration (s) of the milk allergen (s) in whole bovine milk.

In one embodiment, the subject not previously sensitised to one or more bovine milk allergens is a subject not previously directly exposed to one or more bovine milk allergens.

The following embodiments may relate to any of the above aspects

In various embodiments, the composition comprises one or more bovine milk allergens at a concentration at least 50% greater, at least 75% greater, at least 100% greater, at least 200% greater, at least 300% greater, at least 400% greater, at least 500% greater, at least 600% greater, at least 700% greater, at least 800% greater, at least 900% greater, or at least 1000% greater than the concentration(s) of the milk allergen(s) in whole bovine milk. In representative embodiments in which one of the one or more bovine milk allergens is al ha_si -casein, the composition comprises alpha_srcasein at a concentration of at least about 15g/L, at least about 20g/L, at least about 30g/L, at least about 40g/L, at least about 50g/L, at least about 60g/L, at least about 70g/L, at least about 80g/L, at least about 90g/L, at least about lOOg/L.

In representative embodiments in which one of the one or more bovine milk allergens is alpha-lactoalbumin, the composition comprises alpha-lactalbumin at a concentration of at least about 1.5g/L, at least about 2g/L, at least about 3g/L, at least about 4g/L, at least about 5g/L, at least about 6g/L, at least about 7g/L, at least about 8g/L, at least about 9g/L, or at least about 10g/L.

In representative embodiments in which one of the one or more bovine milk allergens is beta lactoglobulin, the composition comprises beta-lactoglobulin at a concentration of at least about 4.5g/L, at least about 6g/L, at least about 9g/L, at least about 12g/L, at least about 15g/L, at least about 18g/L, at least about 21g/L, at least about 24g/L, at least about 27g/L, or at least about 30g/L.

In representative embodiments in which one of the one or more bovine milk allergens is bovine serum albumin, the composition comprises bovine serum albumin at a concentration of at least about 0.6g/L, at least about 0.8g/L, at least about 1.2g/L, at least about 1.6g/L, at least about 2g/L, at least about 2.4g/L, at least about 2.8g/L, at least about 3.2g/L, at least about 3.6g/L, or at least about 4g/L.

In various embodiments, the one or more bovine milk proteins comprise at least about 10% of the subject's recommended protein daily intake.

In various embodiments, the amount sufficient to elicit tolerance is an amount above the concentration range associated with risk of eliciting sensitivity to one or more milk allergens.

In various embodiments, the amount sufficient to elicit tolerance is insufficient to elicit sensitivity. In one embodiment, the amount insufficient to elicit sensitivity is one or more of the following:

a) an amount insufficient to elicit sensitivity to one or more bovine milk allergens as assessed by skin prick test;

b) an amount insufficient to elicit sensitivity to one or more bovine allergens as

assessed by a determination of the presence of bovine milk allergen-specific IgE; c) an amount insufficient to elicit more than 0.35 kilounits of IgE/L as assessed by Pharmacia Diagnositcs UNICAP®, for example by Pharmacia Diagnostics UNICAPIOO®;

d) an amount insufficient to elicit one or more clinical symptoms of milk allergy. In various embodiments, the amount sufficient to elicit tolerance is from about 2g to about 15g per day total milk protein allergen.

In various embodiments, the administration is daily, twice-daily, three times daily, four times daily, or more than four times daily.

In various embodiments, the administration is for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or at least about six months.

In various embodiments, the administration is

a) of an amount of one or more bovine milk allergens sufficient to elicit tolerance to one or more bovine milk allergens,

b) at least daily,

c) for at least one month.

In various embodiments, sensitivity to one or more bovine milk allergens is one or more of the following:

a) sensitivity to one or more bovine milk allergens as assessed by skin prick test;

b) sensitivity to one or more bovine allergens as assessed by a determination of the presence of bovine milk allergen-specific IgE;

c) more than 0.35 kilounits of [bovine milk allergen-specific] IgE/L as assessed by Pharmacia Diagnositcs UNICAP®;

d) presence of one or more clinical symptoms of milk allergy.

In various embodiments, sensitivity to one or more bovine milk allergens is sensitivity to one or more bovine allergens as assessed by a determination of the presence of bovine milk allergen- specific IgGl.

In one embodiment, the one or more milk proteins are administered in the form of a composition with a physiologically acceptable diluent, adjuvant, carrier or excipient.

In one embodiment, said physiologically acceptable diluent, adjuvant, carrier or excipient is a food. In one embodiment, the food is cultured milk, yoghurt, cheese, milk drink or milk powder.

Alternatively the composition is a pharmaceutical composition and said excipient or diluent is pharmaceutically acceptable diluent, adjuvant, carrier or excipient.

In various embodiments, the subject is a foetal, a neonatal, an infant, a child, a juvenile, or an adult subject.

In embodiments where the subject is a foetal subject, the method comprises administering the one or more milk proteins or a composition comprising one or more milk allergens to the foetal subject's mother. It will be appreciated that in such embodiments, the administration to the subject may be considered indirect administration. In one embodiment, the composition is a maternal formula or a maternal supplement.

In certain embodiments where the subject is a neonatal, an infant, or a child subject, the method comprises administering a composition comprising one or more milk allergens to the subject. Again, it will be appreciated that in such embodiments, the administration to the subject may be considered direct administration.

In other embodiments, such as where the subject is a breastfeeding neonatal, infant, or child subject, the method comprises administering the one or more milk allergens or a composition comprising one or more milk allergens to the subject's mother. It will be appreciated that in such embodiments, the administration to the subject may be considered indirect administration.

The composition may be an infant formula, follow-on formula, growing-up formula or dietetic product, including hypoallergenic embodiments of such compositions.

In preferred embodiments where the subject is a juvenile or an adult subject, the method comprises administering a composition comprising one or more milk allergens to the subject. Preferably, the composition is a supplement, formula, dietetic product, drink, or food.

The invention further provides one or more milk allergens for treating or preventing sensitivity to one or more milk allergens and one or more milk allergens in the manufacture of a composition for treating or preventing sensitivity to one or more milk allergens. The composition may be a composition such as those as described below including, for example, a drink, a food or a medicament.

It will be appreciated that the invention also contemplates the use of one or more milk allergens in the manufacture of a composition of the invention, for example a composition for treating or preventing eczema in a subject.

In various embodiments the composition is a unit dosage form.

In one embodiment the composition is suitable for oral administration. In other embodiments, the composition is suitable for parenteral administration. In embodiments relating to preventing eczema in a foetal subject, the composition is suitable for oral administration to a pregnant mother during gestation.

In various embodiments, the sensitivity is or exacerbates one or more of the following conditions: food protein-induced enterocolitis syndrome (FPIES), atoptic dermatitis, eosinophilic esophagitis, colic including infantile colic, isolated failure to thrive, lethargy, cutaneous reactions, including urticaria, angioedema, and pruritus, gastrointestinal symptoms including vomiting and diarrhoea, and respiratory symptoms including chronic rhinitis and recurrent wheezing. In one embodiment, the sensitivity is anaphylaxis. In one embodiment, the sensitivity or adverse immune response is or exacerbates one or more conditions associated with allergy, such as eczema including atopic eczema (also known as infantile eczema, flexural eczema or atopic dermatitis), xerotic eczema (also known as asteatotic eczema), seborrhoeric dermatitis, dyshidrosis, discoid eczema, venous eczema, Duhring's disease, or neurodermatitis, [etc].

In various embodiments, the method of modulating in a subject in need thereof an immune response to one or more bovine milk allergens is downregulation of an existing immune response. Accordingly, the invention contemplates both prophylactic administration of one or more bovine milk allergens, and therapeutic administration of one or more milk allergens, for example to a subject developing or with sensitivity to one or more bovine milk allergens.

In one embodiment the composition or product comprises, consists essentially of, or consists of fresh whole milk, fresh skim milk, recombined or reconstituted whole or skim milk powder, skim milk concentrate, skim milk retentate, concentrated milk, ultrafiltered milk retentate, milk protein concentrate (MPC), milk protein isolate (MP I), calcium depleted milk protein concentrate (MPC), casein, caseinate, colostrum, a colostrum extract, colostrum protein concentrate (CPC), colostrum whey, an immunoglobulin extract from colostrum, whey (including sweet whey, lactic acid whey, mineral acid whey, or reconstituted whey powder), whey protein isolate (WPI), whey protein concentrate (WPC), a composition derived from any milk or colostrum processing stream, a composition derived from the retentate or permeate obtained by ultrafiltration or microfiltration of any milk or colostrum processing stream, a composition derived from the breakthrough or adsorbed extract obtained by chromatographic (including but not limited to ion and gel permeation chromatography) separation of any milk or colostrum processing stream, extracts of any of these milk derivatives including extracts prepared by multistage fractionation, solvent fractionation, supercritical fractionation, near critical fractionation, distillation, centrifugal fractionation, hydrolysates of any of these materials, extracts of the hydrolysates, and any combination of any two or more of these materials, including combinations of hydrolysed and/or non-hydrolysed extracts. It should be understood that the source of these materials may be milk or colostrum or a combination thereof.

In one embodiment, the composition is a dairy product, for example a milk drink, comprising an added source of milk protein or milk allergen.

As used herein the term "and/ or" means "and" or "or", or both.

As used herein "(s)" following a noun means the plural and/or singular forms of the noun. It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly d sclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing splenocyte responsiveness to BLG (5mg/mL) by animals fed BLG for

30 days from 21 days of age, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 2 is a graph showing splenocyte responsiveness to BLG by animals fed BLG for 30 days from 56 days of age, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 3 is a graph showing levels of IgGl in serum of 21 day old mice fed BLG for 30 days, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 4 is a graph showing levels of IgGl in serum of 56 day old mice fed BLG for 30 days, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 5 is a graph showing levels of IgG2a in serum of 21 day old mice fed BLG for 30 days, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 6 is a graph showing levels of IgG2a in serum of 56 day old mice fed BLG for 30 days, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 7 is a graph showing levels of IgE in serum of 21 day old mice fed BLG for 30 days, as described in Example 1 herein. Data shown are averages of 20 mice per group.

Figure 8 is a graph showing levels of IgE in serum of 56 day old mice fed BLG for 30 days, as described in Example 1 herein. Data shown are averages of 20 mice per group. DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

The term "bovine whole milk" and grammatical equivalents thereof contemplates whole milk from bovine fed non-modified feedstock, preferably having one or more of the following:

approximately lOg/L alpha_si -casein, approximately g/L alpha-lactalbumin, approximately 9g/L beta-lactoglobulin, approximately 0.4g/L bovine serum albumin, or approximately O.lg/L lactoferrin.

The term "comprising" as used in this specification means "consisting at least in part of.

When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement or claim, all need to be present but other features can also be present.

Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

The term "treat" and its derivatives should be interpreted in their broadest possible context.

The term should not be taken to imply that a subject is treated until total recovery. Accordingly,

"treat" broadly includes maintaining a subject's d sease progression or symptoms at a substantially static level, increasing a subject's rate of recovery, amelioration and/ or prevention of the onset of the symptoms or severity of a particular condition, or extending a patient's quality of life. The term

"treat" also broadly includes the maintenance of good health for sensitive individuals and building stamina for disease prevention.

The term "dietetic product" means a product specially processed or formulated to satisfy particular dietary requirements which exist because of a particular physical or physiological condition and/ or specific diseases and disorders and which are presented as such.

The term "milk allergens" as used in the specification contemplates one or more components of milk capable of eliciting sensitivity or an adverse immune response to bovine milk, and includes one or more milk proteins and one or more milk protein fragments, including one or more allergenic peptides derived from a milk protein. Exemplary milk allergans can be selected from or derived from alp ha_si -casein, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, or lactoferrin.

The term "oral administration" includes oral, buccal, enteral and intra-gastric administration.

The term "pharmaceutically acceptable carrier" is intended to refer to a carrier including but not limited to an excipient, diluent, auxiliary or combination thereof that can be administered to a subject as a component of a composition of the invention that does not reduce the activity of the composition and is not toxic when administered in doses sufficient to deliver an effective amount of the active ingredient. The formulations can be administered orally, nasally and topically. A "subject" is an animal, preferably a mammal, more preferably a mammalian companion animal or human. Preferred companion animals include cats, dogs and horses. In one embodiment the subject is a human. Preferably the human is an adult, a child, or an infant.

An "effective amount" is the amount required to confer therapeutic effect. The

interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, et al. (1966). Body surface area can be approximately determined from height and weight of the subject. See, e.g., Scientific Tables, Geigy

Pharmaceuticals, Ardley, New York, 1970, 537. Effective doses also vary, as recognized by those skilled in the art, dependent on route of administration, carrier usage, and the like.

The term "tolerizing amount" as used in the specification with reference to one or more milk allergens means an amount of milk allergen able to be administered to the subject without sensitising the subject to that particular milk allergen. In some embodiments, the tolerizing amount is less than a sensitising amount. In other embodiments, the tolerizing amount is more than a sensitizing amount.

2. Milk allergens

Most common food allergies are proteinaceous— typically proteins or fragments of proteins that are resistant to digestion which are recognised by IgE immunoglobulins and promote an allergic response. Potentially allergenic proteins present in bovine milk include the following (noting that fragments of same are also potentially allergenic).

Alpha_srcasein is a known milk allergen (11-15 g/L). It is one of the four major caseins, it is a

27-32 kDa molecule and accounts for 40% of the casein fraction of bovine milk. It is important in the transport of the other casein variants from the endoplasmic reticulum to the Golgi apparatus. It has been found that primarily intact alpha_si-casein or larger IgE-reactive portions are responsible for the IgE-mediated symptoms of food allergy.

Alpha-lactalbumin is a 14kDa whey protein found in bovine milk (~ lg/L) as well as in the milk of many other mammalian species. It differs from beta-lactoglobulin in that it lacks any free thiol groups that can act as a starting point for covalent aggregation reactions. It is known to increase the production of lactose in primates. As a monomer, alpha-lactalbumin has been found to bind calcium and zinc ions, and may act as a potential antibacterial or antitumor properties.

Beta-lactoglobulin is a major whey protein found in bovine milk (~3 g/L) as a well as being present in the milk of other mammahan species. Beta-lactoglobulin is a small 18 kDa protein with 162 residues and is predominately dimeric in physiological conditions. No clear function has been determined for beta-lactoglobulin but there is strong suggestion that it is associated with transport or exists primarily as a food source. Bovine serum albumin is a serum albumin protein with a molecular weight of 66.4 kDa. It is used in many applications as it is stable, has a low reactivity in many biochemical reactions, large quantities readily available and cost-effective. It has found use as a carrier protein, enzyme stabiliser, Northern and Southern and dot blot hybridizations and as a blocking agent.

Lactoferrin is a multifunctional protein of the transferring family. It is a 80 kDa globular glycoprotein and is present in many secretary fluids such as milk, saliva and tears. It is found in very high levels in human and bovine milk. Lactoferrin is a component of the immune system and is known to have antibacterial, antiviral and antifungal activity. This antimicrobial activity is attributed to its ability to interact with the cells walls of bacteria, viruses and fungi.

3. Types of allergic response

The symptoms of food allergies are varied, and may present as food protein-induced enterocolitis syndrome (FPIES), atoptic dermatitis, eosinophilic esophagitis, colic including infantile colic, isolated failure to thrive, lethargy, cutaneous reactions, including urticaria, angioedema, and pruritus, gastrointestinal symptoms including vomiting and diarrhoea, and respiratory symptoms including chronic rhinitis and recurrent wheezing. In severe cases, anaphylaxis may manifest.

In various exemplary embodiments, the sensitivity to one or more bovine milk allergens exacerbates one or more adverse immunological conditions, such as one or more conditions associated with allergy. In one embodiment the condition is asthma. In one embodiment the condition is an atopic condition. In yet another embodiment the condition is an eosinophilia.

In one embodiment the condition is selected from allergic rhinitis, hay fever, atopic rhinoconjunctivitis, urticaria, asthma and atopic eczema.

In one embodiment the condition is selected from contact dermatitis, eczema, hives

(urticaria), allergic conjunctivitis, hay fever, allergic rhinitis, airborne allergies including tree (e.g. birch pollen), weed (e.g. ragweed), and grass pollen allergies, latex allergies, other food allergies (e.g. peanut, shellfish), drug allergies (e.g. to penicillin), insect sting allergies (e.g. honeybee allergies, wasp allergies, hornet allergies, yellow jacket allergies, fire ant allergies), mold allergies (e.g. to alternaria, cladosporium, aspergillus, penicillium, helminthosporium, epicoccum, fusarium, mucor, rhizopus, and aureobasidium), dust mite allergies, animal allergies (e.g. household pets such as cats and dogs), allergic bronchopulmonary aspergillosis, occupational asthma, and episodic angioedema with eosinophilia.

In one embodiment the allergic condition is selected from airway, lung, blood or skin eosinophilia. In another embodiment, the eosinophilia is selected from eosinophilic ascites, eosinophilic cellulitis, eosinophilic fasciitis, eosinophilic gastroenteritis, coeliac disease, allergic colitis, eosinophilic esophagitis, eosinophilic pancreatitis, eosinophilic pneumonias, bronchiectasis, eosinophilic synovitis, nasal eosinophilia, tropical pulmonary eosinophilia, Churg Strauss syndrome, pulmonary eosinophilia, idiopathic hyper-eosinophihc syndrome, inflammatory bowel disease, eosinophilic cholangitis, eosinophilic leukaemia and other eosinophilic cancers, familial (hereditary eosinophilia), eosinophilic granuloma, eosinophilia-myalgia syndrome, cystic fibrosis, nasal polyposis, eosinophil meningitis, Wegener's granulomatosis, polyarteritis nodosa, pemphigus vulgaris, bullous pemphigoid, dermatitis herpetiformis, erythema multiforme, eosinophilic cellulites, parasitic infections (Ascaris Toxocara canis, Filariasis, Ankylostomiasis, Trichinosis,

Strongvloidiasis, Fascioliasis, Schistosomiasis).

In one embodiment the subject is susceptible to anaphylaxis. For example, in one

embodiment the administration of the lactic acid whey or a derivative thereof is prophylactic administration.

4. Compositions useful according to invention

A composition useful herein may be formulated as a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical. Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.

In one embodiment the present invention relates to use of milk allergen or a milk allergen analogue in the manufacture of a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical. Preferably the composition is formulated for oral administration. Preferably the composition is formulated for oral or parenteral administration. Preferably the composition is for decreasing a subject's risk of developing sensitivity to one or more bovine milk allergens, the method comprising orally administering to the subject one or more bovine milk allergens, wherein the administration is of a composition comprising one or more bovine milk allergens wherein the one or more bovine milk allergens is present at a concentration at least 50% greater than the concentration (s) of the milk allergen(s) in whole bovine milk; and optionally wherein the administration is one or more of the following:

a) of an amount sufficient to elicit tolerance to one or more bovine milk allergens; or b) of a frequency sufficient to elicit tolerance to one or more bovine milk allergens; or c) of a duration sufficient to elicit tolerance to one or more bovine milk allergens; or d) commences prior to any previous direct exposure of the subject to bovine milk or one or more bovine milk allergens (such that the administration of the one or more milk allergens represents the first direct exposure of the subject to the one or more milk allergens); or

e) commences prior to established sensitivity to one or more bovine milk allergens; or f) any combination of two or more of a) to e), in a subject in need thereof, or other uses, as described above.

In one embodiment the composition is in the form of a tablet, a caplet, a pill, a hard or soft capsule or a lozenge.

In one embodiment the composition is in the form of a cachet, a dispensable powder, granules, a suspension, an elixir, a hquid, a drink, or any other form that can be added to food or drink, including for example water or fruit juice. In one embodiment the composition is an enteral product, a solid enteral product or a liquid enteral product.

In one embodiment the composition further comprises one or more constituents (such as antioxidants) which prevent or reduce degradation of the composition during storage or after administration.

In one embodiment, compositions useful herein include any edible consumer product which is able to carry protein. When the composition comprises as one or more additional therapeutic agent a fat or lipid-based factor, such as milk fat, the edible consumer product is one able to carry fats, salts, acids or lipids. Examples of suitable edible consumer products include baked goods, powders, hquids, confectionary products, reconstituted fruit products, snack bars, food bards muesli bars, spreads, sauces, dips, dairy products including ice creams, yoghurts and cheeses, drinks including dairy and non-dairy based drinks (such as milk drinks including milk shakes, and yogurt drinks), milk powders, sports supplements including dairy and non-dairy based sports supplements, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets. Within this embodiment, a composition useful herein may also be an infant formula, in powder or Hquid form. Suitable nutraceutical compositions useful herein may be provided in similar forms.

Compositions useful herein may further include other factors such as calcium, zinc, magnesium, selenium, vitamin C, vitamin D, vitamin E, vitamin K2, complex carbohydrates, edible or cooking oils including palm, olive, soybean, canola, corn, sunflower, safflower, peanut, grape seed, sesame, nut, almond, cashew, hazelnut, macadamia, pecan, pistachio, and walnut, and other edibles include acai, amaranth, apricot, argan, artichoke, avocado, babassu, ben, blackcurrant seed, borage seed, borneo tallow nut, bottle gourd, buffalo gourd, carob pod (algaroba), cohune, coriander seed, evening primrose, false flax, hemp, kapok seed, lallemantia, meadowfoam seed, mustard, okra seed (hibiscus seed), perilla seed, pequi, pine nut, poppyseed, prune kernel, pumpkin seed, quinoa, ramtil, rice bran, tea (camellia), thistle, watermelon seed, or wheat germ oil, or a combination thereof.

The compositions useful herein are formulated to allow for oral administration. In general, for oral administration a dietary (a food, food additive or food supplement for example), nutraceutical or pharmaceutical composition useful herein may be formulated by a skilled worker according to known formulation techniques.

Thus, a pharmaceutical composition useful according to the invention may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected with regard to the intended route of administration and standard pharmaceutical practice. See for example, Remington 's Pharmaceutical Sciences, 16th edition, Osol, A. Ed., Mack Publishing Co., 1980.

While the preferred route of administration is oral, it should be understood that any mode of administration may be suitable for any composition of the invention, including administration by multiple routes, including different routes for different agents. Therefore, rectal administration of any composition of the invention is also contemplated. Administration of one or more milk allergens, optionally with at least one additional therapeutic factor, by a first administration route accompanied by separate, simultaneous or sequential administration by a second administration route is also contemplated; for example, oral administration of one or more milk allergens accompanied by rectal administration of another milk allergen or of an additional therapeutic agent.

A dosage form useful herein may be administered orally as a powder, liquid, tablet or capsule. Suitable dosage forms may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, or have an enteric coating. Suitable enteric coatings are known. Enteric coatings surrounding the active ingredients and prevent the release of the active ingredients in the stomach but allow release after the dosage form has left the stomach. Dosage forms useful herein may be adapted for immediate, delayed, modified, sustained, pulsed or controlled release of the active components. Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents.

Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. Active ingredients can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tabletting agent. Pharmaceutical compositions can also be administered via the parenteral route.

Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Cyclodextrins, or other solubilising agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent. Sustained-release preparations may be prepared incorporating one or more milk allergens, optionally with at least one additional therapeutic factor. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing milk allergens, and when present the at least one additional therapeutic agent. The matrices may be in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (see US 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non- degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers.

The efficacy of a composition useful according to the invention can be evaluated both in vitro and in vivo. See, e.g., the examples below. Briefly, in one embodiment the composition can be tested for its ability, to for example, tolerize in vitro. For in vivo studies, the composition can be fed to an animal (e.g., a mouse) and its allergenic effects then assessed. Based on the results, an appropriate dosage range and administration route can be determined.

The compositions useful herein may be used alone or in combination with one or more other therapeutic agents. The therapeutic agent may be a food, drink, food additive, drink additive, food component, drink component, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical. The therapeutic agent is preferably effective to attenuate one or more of the symptoms of a condition associated with allergy.

In one embodiment the milk allergen or a milk allergen analogue, optionally with at least one additional therapeutic agent are formulated for administration separately, simultaneously or sequentially with at least one anti- allergenic agent or anti-allergy therapy described herein.

In one embodiment the milk allergen or a milk allergen analogue, optionally with at least one additional therapeutic agent are formulated for coadministration with the at least one anti- allergenic agent or anti-allergy therapy described herein.

In one embodiment the milk allergen or a milk allergen analogue, optionally with at least one additional therapeutic agent are formulated for sequential administration with the at least one anti- allergenic agent or anti-allergy therapy described herein.

When used in combination with another therapeutic agent, the administration of a composition useful herein and the other therapeutic agent may be simultaneous or sequential.

Simultaneous administration includes the administration of a single dosage form that comprises all components or the administration of separate dosage forms at substantially the same time.

Sequential administration includes administration according to different schedules, preferably so that there is an overlap in the periods during which the composition useful herein and other therapeutic agent are provided. Suitable agents with which the compositions of the invention can be co-administered include immunosuppressants, immunotherapeutic agents, and other anti-allergy agents known in the art. Such agents are preferably administered orally, or by rectal administration.

Additionally, it is contemplated that a composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a subject in particular instances. For example, therapeutic agents that target the same or different facets of the disease process may be used.

In one embodiment, a composition useful herein includes or is administered simultaneously or sequentially with other milk components such as milk fat, whey proteins, whey protein fractions (including acidic or basic whey protein fractions or a combination thereof), glycomacropeptide, lactoferrin, vitamin D or calcium, or combinations thereof. Useful milk component-containing compositions include compositions such as a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food or nutraceutical. Milk fractions enriched for these components may also be employed.

As will be appreciated, the dose of the composition administered, the period of

administration, and the general administration regime may differ between subjects depending on such variables as the severity of symptoms of a subject, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/ or general health of a subject. However, by way of general example, the inventors contemplate administration of from about 1 mg to about 2000 mg per kg body weight of a composition useful herein is administered per day, preferably 1 mg to about lOOOmg per kg per day, preferably about 50 to about 500 mg per kg per day. In one embodiment, the inventors contemplate administration of from about 0.05 mg to about 250 mg per kg body weight of a pharmaceutical composition useful herein.

It should be appreciated that administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate. It should be understood that a person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine an effective dosage regime (including daily dose and timing of administration) for a given condition.

The present invention also relates to a dietary, nutraceutical or oral pharmaceutical composition comprising, consisting essentially of or consisting of milk allergen or a milk allergen analogue. Preferably the composition consists essentially of about 0.1 to 99 wt % milk allergen or a milk allergen analogue. More preferably the composition consists essentially of about 0.5 to 10 wt % milk allergen. Most preferably the composition consists essentially of about 1 wt % milk allergen and about 5 wt % . In one embodiment a composition of the invention is a milk fraction, preferably a milk protein fraction. In one embodiment the milk fraction comprises at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% by weight milk protein, and useful ranges may be selected from any of these values (for example, from about 1 to about 99% by weight, from about 5 to about 99% by weight, from about 10 to about 99% by weight, from about 15 to about 99% by weight, from about 20 to about 99% by weight, from about 25 to about 99% by weight, from about 30 to about 99% by weight, from about 35 to about 99% by weight, from about 40 to about 99% by weight, from about 45 to about 99% by weight, from about 50 to about 99% by weight, from about 55 to about 99% by weight, from about 60 to about 99% by weight, from about 65 to about 99% by weight, from about 70 to about 99% by weight, from about 75 to about 99% by weight, from about 80 to about 99% by weight, from about 85 to about 99% by weight, from about 90 to about 99% by weight, or from about 95 to about 99% by weight). Preferably, when the composition comprises at least one additional therapeutic agent, the composition may additionally comprise a milk fat fraction.

In one embodiment the milk allergen or a milk allergen analogue is administered daily for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more weeks, including for such duration before exposure of the subject to another source of milk allergen.

In one embodiment the milk allergen or a milk allergen analogue is administered for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or for at least about 1, 2, 3, 4, 5, 6, 7 or 8 weeks or for at least about 1, 2, 3, 4, 5 or 6 months, for example for such duration before exposure of the subject to another source of milk allergen.

Preferably the milk allergen or a milk allergen analogue is administered at least once daily including continuously over a day orally.

In various embodiments, the composition comprises one or more bovine milk allergens at a concentration at least 50% greater, at least 75% greater, at least 100% greater, at least 200% greater, at least 300% greater, at least 400% greater, at least 500% greater, at least 600% greater, at least 700% greater, at least 800% greater, at least 900% greater, or at least 1000% greater than the concentration(s) of the milk allergen(s) in whole bovine milk.

When one or more bovine milk allergens is alpha_si-casein, the composition comprises alpha_si- casein at a concentration of at least about 15g/L, at least about 20g/L, at least about 30g/L, at least about 40g/L, at least about 50g/L, at least about 60g/L, at least about 70g/L, at least about 80g/L, at least about 90g/L, at least about lOOg/L.

When one or more bovine milk allergens is alpha-lactoalbumin, the composition comprises alpha-lactalbumin at a concentration of at least about 1.5g/L, at least about 2g/L, at least about 3g/L, at least about 4g/L, at least about 5g/L, at least about 6g/L, at least about 7g/L, at least about 8g/L, at least about 9g/L, or at least about Og/L.

When one or more bovine milk allergens is beta-lactoglobulin, the composition comprises beta-lactoglobulin at a concentration of at least about 4.5g/L, at least about 6g/L, at least about 9g/L, at least about 12g/L, at least about 5g/L, at least about 8g/L, at least about 21g/L, at least about 24g/L, at least about 27g/L, or at least about 30g/L.

When one or more bovine milk allergens is bovine serum albumin, the composition comprises bovine serum albumin at a concentration of at least about 0.6g/L, at least about 0.8g/L, at least about 1.2g/L, at least about 1.6g/L, at least about 2g/L, at least about 2.4g/L, at least about 2.8g/L, at least about 3.2g/L, at least about 3.6g/L, or at least about 4g/L.

Milk allergens of the prevent invention can be added to food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, enteral or parenteral feeding product, meal replacement, cosmeceutical, any edible consumer product that is able to carry protein, or pharmaceutical. Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.

Examples of suitable edible consumer products include powders, liquids, confectionary products including chocolate, gels, ice creams, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy products including yoghurts and cheeses, drinks including dairy and non-dairy based drinks (such as milk drinks and yogurt drinks), milk powders, sports supplements including dairy and non-dairy based sports supplements, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets. Suitable nutraceutical compositions useful herein may be provided in similar forms.

Further examples of dosage forms of the invention include, but are not limited to modified- release (MR) dosage forms including delayed-release (DR) forms; prolonged-action (PA) forms; controlled-release (CR) forms; extended-release (ER) forms; timed-release (TR) forms; and long- acting (LA) forms. These formulations effect delayed total allergen release for some time after administration, and/or release in small aliquots intermittendy after administration, and/ or release slowly at a controlled rate governed by the delivery system, and/ or release at a constant rate that does not vary, and/ or release for a significantly longer period than usual formulations.

5. Dosage

As will be appreciated, the dose of the composition administered, the period of

administration, and the general administration regime may differ between subjects depending on such variables as the amount of milk allergen required to sensitise a subject, the type of milk allergen, and the age, sex and/ or general health of a subject. However, by way of general example, the inventors contemplate administration of from about 1 mg to about 2000 mg per kg body weight of a milk fat extract useful herein per day, preferably 1 mg to about lOOOmg per kg per day, preferably about 1 to about 500 mg/kg/ day, alternatively about 150 to about 410 mg/kg/day, alternatively about 1 to about 100 mg/kg/ day or about 1 to about 20 mg/kg/ day.

It should be appreciated that administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate.

Each dosage can vary depending on the subject's reaction to the previous dosage. For example, a subsequent dosage could be less than the initial dosage if the subject is starting to display clinical symptoms of milk allergies.

In various embodiments, the administration is for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about 12 months or at least as long as what is required for a subject to become tolerant to milk allergens.

It should be understood that a person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine an effective dosage regime (including daily dose and timing of administration) for a given condition.

In one embodiment the composition is formulated for separate, simultaneous or sequential administration with a food. In one embodiment the food is selected from confectionary, milk, milk product, milk powder, reconstituted milk, cultured milk, yoghurt, drinking yoghurt, set yoghurt, beverage, food additive, drink additive, dietary supplement, nutritional product, medical food, medicament, nutraceutical or pharmaceutical.

Simultaneous administration includes the administration of a single dosage form that comprises all components or the administration of separate dosage forms at substantially the same time. Sequential administration includes administration according to different schedules, preferably so that there is an overlap in the periods during which the composition useful herein and other therapeutic agent are provided.

Additionally, it is contemplated that a composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a subject in particular instances. For example, therapeutic agents that target the same or different facets of the desired outcome may be used.

In one embodiment the additional active ingredients may include lipids, carbohydrates, other proteins, minerals or vitamins, or other components. 6. Determining sensitivity

In one embodiment of the present invention includes the administration of a milk allergen in an amount to tolerize but not sensitise the subject. This amount can be determined by various methods known to a person skilled in the art, these included but are not limited to the skin prick test, determining the presence of bovine milk allergen-specific IgE, UNICAP® 100 diagnostics and the presence of clinical milk allergy symptoms.

The skin prick test is a simple diagnostic test of allergies where the aim is to provoke a controlled allergic response. The test is as simple as pricking a few drops of purified allergen onto the subjects skin surface then an allergic reaction, if any, if monitored. A tolerisable dosage will not provoke any allergic reaction.

Another common method is to determine the presence of bovine milk allergen-specific IgE using method such as radioallergosorbent (RAST) based allergy diagnosis.

UniCAP® 100 is another method that is in vitro and requires a subjects blood sample. The blood sample is used to test various allergens at the same time resulting in a quick and accurate test. The three main UniCAP test are the total IgE, phadiatop and specific IgE test. UniCAP® 100 is also considered safer as it is done in vitro rather than subjecting an individual to an allergic response.

Another method is the presence of clinical symptoms of milk allergy. These include skin rash, hives, vomiting, gastric distress, anaphylactic reactions, atopic dermatitis, wheeze, infantile colic, gastroesophageal reflux (GER), oesophagitis, allergic colitis headache/migraine, oral irritation, and constipation. These symptoms can be immediate or delayed.

7. Protein intake

Protein is an important part of a balanced diet for a mammal. Proteins provide essential amino acids and polypeptides that the body requires to sustain itself and grow. The recommended intake of protein varies between individuals. Recommended intake is dependent on many factors such as age, height, weight, gender, an individual's metabolism or how active an individual is.

For example, a woman's requirement for protein increases during pregnancy in order to nourish her child, as does that of an individual healing from an injury, or that of a very active individual.

There is still debate as to the recommended dally intake of protein. However, in general, the recommended intake for protein for an average person is suggested to be 10% to 25 % of an individual's caloric intake or 0.8 g/kg to 1.8 g/kg/day.

On the other hand, excess protein is also detrimental to an individual's health. It has been suggested that excess protein intake results in an individual's kidneys being over worked. It has also been suggested that excessive protein intake results in an increased level of calcium being excreted from the body. Therefore it is important that protein intake for subjects administered the formulation of the present invention is maintained.

If a subject has an adequate protein intake before treatment, the formulation of the present application may be used to substitute part or all of that intake. However, if an individual's usual intake is lower than the recommended intake, the formulation of the present application may be administered so as to augment the subject's usual protein intake in order to reach a recommended protein intake.

In the present invention, a non-sensitizing amount of milk allergens can be administered to a subject in combination with their usual intake of protein or a non-sensitizing amount of milk allergens can be substituted for a proportion of the subject's usual intake of protein. Protein deficiency is also linked to reduced intelligence or mental retardation. Protein deficiency is generally caused by lack of total food energy as opposed to an individual lacking in solely protein.

Accordingly, in one embodiment of the present invention, at least proportion of an individual's daily protein intake is substituted with milk allergens.

In one embodiment, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,

99, 99.5 or 99.9% of an individual's protein RDI is substitute with milk allergens as described in the present application. Useful ranges may be selected between any of these values (for example, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, about 45 to about 99%, about 50 to about 99%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% by weight.

In another embodiment of the present invention, the formulation of the present invention is administered on top of an individual's existing protein intake, about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 99.9% as a percentage of an individual's usual protein intake is supplemented on top of an individual's usual protein intake with the formulation of the present invention. Useful ranges may be selected between any of these values (for example, about 0.5 to about 95%, about 5 to about 95%, about 10 to about 95%, about 15 to about 95%, about 20 to about 95%, about 25 to about 95%, about 30 to about 95%, about 35 to about 95%, about 40 to about 95%, about 45 to about 95%, about 50 to about 95%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, about 45 to about 99%, about 50 to about 99%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, and about 50 to about 70% by weight.

The amount of one or more milk allergens substituted into an individual's protein RDI is de endent on the amount of milk allergen that can be administered to that individual without the risk of sensitization. Methods of determining sensitization are described herein and are well-known to a person skilled in the art.

In one embodiment the one or more milk allergens is administered orally.

In one embodiment the one or more milk allergens, optionally with at least one additional therapeutic agent, is administered daily for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks, for example including for such duration before the exposure of the subject to milk or another source of milk allergen.

In one embodiment the one or more milk allergens, optionally with at least one additional therapeutic agent, is administered for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or for at least about 1, 2, 3, 4, 5, 6, 7 or 8 weeks or for at least about 1, 2, 3, 4, 5 or 6 months, for example including for such duration before the exposure of the subject to milk or another source of milk allergen.

Preferably the one or more milk allergens, optionally with at least one additional therapeutic agent, is administered at least once daily (including continuously over a day) orally, or by a combination of administrative routes (oral and rectal, for example).

EXAMPLES

1. Example 1— Mouse model

Introduction

The development of allergy (or hypersensitivity) to cow's milk induces several immune parameters that can be measured. In particular, individuals will make high levels of immune hormones (cytokines) such as interleukin 5 (IL-5) and interleukin 4 (IL-4), induce production of mast-cell protease (MMP) and the production of a specific isotype of antibody IgE. This study investigated the potential of ameliorating an allergenic response in animals by feeding different concentrations of beta-lactoglobulin (BLG), the most allergenic protein present in cow's milk, for 30 days to different age groups of mice, and monitoring the induction of these immune parameters. Methods

Groups of twenty C57B16 mice aged 21 or 56 days were fed BLG for 30 days. Groups were as follows:

. PBS fed mice (control)

2. Mice injected IP with PBS/alum (control)

3. Mice injected IP with BLG/ alum (control)

4. Mice injected IP with BLG/ alum and fed lOx BLG from day of IP

5. Mice fed daily for 30 days lx, lOx, lOOx the concentration of BLG found in cow's milk (3g/L). The 56 day old mice were fed only the lOx concentration.

Mice were sacrificed and heart bled. Serum was collected for analysis for IgGl , IgG2a, IgE.

Mice were sacrificed and animal weights were recorded prior to spleens being collected.

Splenocytes were isolated using the Total Murine Splenocyte Preparation Method and plated at a concentration of 2 x 10⁶ cells /ml in 96 well tissue culture plates (Falcon MicroTest®) prior to being stimulated with the mitogen Concanavalin A (T cell compartment— Sigma ensiformis Jack Bean Type IV) at 1.25 μg/ml or BLG (5mg/ml). Plates were incubated for 72 hours in a 5% C02 incubator, Thymidine [6-₃H] - (14.4Ci/mmol (0.533 TBq/mmol) Amersham) was added to the wells and the plate incubated for a further 16 hours. Plates were harvested onto filter membranes - glass fibre printed Filtermat A for 1450 MicroBeta™ (Perkin Elmer) using a Tomtec cell harvester.

Filtermats were placed into Sample bags for Microbeta™ (Perkin Elmer) and 4 mis of Betaplate Scint fluid (Perkin Elmer) was added prior to plates being read in a Wallac 1 50 MicroBeta Plus Liquid scintillation counter.

Results

Splenocytes from mice fed BLG were not responsive to stimulation with BLG. In contrast, the control animals injected IP with BLG/Alum were responsive to stimulation (Figures 1 and 2). As can be seen in Figures 1 and 2, this was observed for both age groups of mice. Animals injected IP with BLG and alum and then fed BLG showed a reduction in splenocyte stimulation compared to those injected IP and not fed BLG.

Elevated IgE and IgGl levels, and essentially unchanged IgG2a levels, in animals challenged IP with BLG and alum were consistent with an allergenic response (Figures 3—8). There was no indication of an allergenic response as indicated by measurement of IgE and IgGl, and of IgG2a, in animals fed lx, lOx or lOOx BLG (Figures 3—8). Animals fed lOx BLG for 30 days following sensitisation resulted in a reduction in the antibodies measured for both age groups of mice, but this reduction was more pronounced in the animals fed BLG for 30 days from the age of 21 days. Discussion

The results from this study indicate that the allergenic effects of exposure to BLG can be reduced by feeding animals tolerising doses daily for 30 days. This is evidenced by the lack of IgGl and IgE in the serum of these animals compared to the allergy (non-tolerised) control (BLG plus alum IP).

These results also demonstrate that it was possible to reduce the allergenic effects in sensitized animals by feeding them lOx BLG for 30 days from the day of sensitization. This feeding regime resulted in a reduction in the levels of both IgE and IgGl. This result was observed in both age groups, but was more pronounced in the younger animals and may indicate that either a higher dose is needed in the older group or they may need to be fed for a longer period of time in order to achieve the same degree of reduction.

This data indicates that the immune system of mammalian subjects can be tolerised to bovine milk allergens by administration of the allergen, for example as the first exposure to the allergen.

2. Example 2— Infant mouse model

This example describes an investigation into the efficacy of administering milk allergens to infant mice as a method of tolerising a subject to milk allergens.

Experimental protocol

7, 14, 21 day old mice are fed milk (supplemented with high quantities of milk allergens as indicated) daily for 1 month. Control animals are fed either milk or PBS. After the treatment period, the mice are then administered a dose of allergen that is sufficient to elicit an allergic response. Tolerance to milk allergens is measured at the end of the experiment. Tolerance is determined by lack of specific IgE production, MMP-1 production and specific activation of T cells against milk allergens, and severity of clinical symptoms.

Sample Treatment

Control 1 7 day old mice - Milk or PBS

#1 Milk + 10-fold increased milk protein mixture

#2 Milk + 20-fold increased milk protein mixture

#3 Milk + 100-fold increased milk protein mixture

#4 Milk + 10-fold increased alpha_sl-casein

#5 Milk + 20-fold increased alpha_si-casein

#6 Milk + 100-fold increased alpha_si-casein

#7 Milk + 10-fold increased alpha- lactalbumin

#8 Milk + 20-fold increased alpha-lactalbumin #9 Milk + 100-fold increased alpha-lactalbumin

#10 Milk + 10-fold increased beta-lactoglobulin

#11 Milk + 20-fold increased beta-lactoglobulin

#12 Milk + 100-fold increased beta-lactoglobulin

#13 Milk + 10-fold increased BSA

#14 Milk + 20-fold increased BSA

#15 Milk + 100-fold increased BSA

#16 Milk + 10-fold increased lactoferrin

#17 Milk + 20-fold increased lactoferrin

#18 Milk + 100-fold increased lactoferrin

Sensitisation

After the treatment period, the mice are administered an amount of milk or allergen that is sufficient to elicit an allergic response. The mice are monitored for any behavioural and physical changes. The severity and duration of the allergic response is recorded for each of the sample groups.

Immunological assay

Blood is collected from each group and an immunological profile is determined using UNICAP®100 analysis.

Results

Results showing no reaction or a less severe reaction amongst individuals of one or more trial cohorts when administered an amount of allergen sufficient to elicit an allergic response in the control group are indicative that the individual(s) of the trial cohort(s) have been tolerised, and thus of the efficacy of the treatment regimen. For example, results including an immunological profile amongst a trial cohort indicative of a lack of specific IgE production, reduced MMP-1 production, and no specific activation of T cells against milk allergens, are indicative of tolerisation of said trial cohort.

Furthermore, results showing cohorts administered higher doses of milk allergens have a more desirable immunological profile and less severe clinical symptoms when compared to the control cohort and lower dose cohort are indicative of the efficacy of the high dose treatment regimen. 3. Example 3 - Various dosage regime

This example describes an investigation of the efficacy of administering multiple doses of allergens compared to a single dose of the same amount of allergen.

Experimental protocol

7, 4, 21 day old mice are fed milk (supplemented with high quantities of milk allergens as indicated) daily for 1 month. Control animals are fed either milk or PBS. After the treatment period, the mice are then administered a dose of allergen that is sufficient to elicit an allergic response. Tolerance to milk allergens is measured at the end of the experiment. Tolerance is determined by lack of specific IgE production, MMP-1 production and specific activation of T cells against milk allergens, and severity of clinical symptoms.

Groups of mice will be fed according to different regimes. However, the total amount of allergens administered each day will stay the same.

Sample Treatment Frequency per day

Control 1 Milk 1

Control 2 Milk 2

Control 3 Milk 3

Sample #1 Milk + 20-fold increased alpha_si -casein 1

Sample #2 Milk + 20-fold increased alpha_sr -casein 2

Sample #3 Milk + 20-fold increased alpha_si -casein 3

Sensitization and immunological assay

The groups are sensitised and subsequent observation and immunological profile recorded as in Example 2.

Results

Results showing no reaction or a less severe reaction amongst individuals of one or more multiple dose trial cohorts when administered an amount of allergen sufficient to elicit an allergic response in the control group are indicative that the individual(s) of the trial cohort(s) have been tolerised, and thus of the efficacy of the frequent administration treatment regimen. For example, results including an immunological profile amongst a multiple dose trial cohort indicative of a lack of specific IgE production, reduced MMP-1 production, and no specific activation of T cells against milk allergens, are indicative of tolerisation of the multiple dose trial cohort.

4. Example 4 - Adult mouse model

This example describes an investigation into the efficacy of administering milk allergens to adult mice as a method of tolerising a subject to milk allergens.

Experimental protocol

8 week old mice are divided into the same cohorts as shown in example 2 above, and are fed milk (supplemented with high quantities of milk allergens as indicated) daily for 1 month. Control animals are fed either milk or PBS. After the treatment period, the mice are then administered a dose of allergen that is sufficient to elicit an allergic response. Tolerance to milk allergens is measured at the end of the experiment. Tolerance will be determined by lack of specific IgE production, MMP-1 production and specific activation of T cells against milk allergens, and severity of clinical symptoms.

Sensitisation and immunological assay

Results

Results showing no reaction or a less severe reaction amongst adult individuals of one or more trial cohorts when administered an amount of allergen sufficient to elicit an allergic response in the control group are indicative that the individual(s) of the trial cohort(s) have been tolerised, and thus of the efficacy of the treatment regimen. For example, results including an immunological profile amongst a trial cohort indicative of a lack of specific IgE production, reduced MMP-1 production, and no specific activation of T cells against milk allergens, are indicative of tolerisation of said adult trial cohort.

Furthermore, results showing cohorts administered higher doses of milk allergens have a more desirable immunological profile and less severe clinical symptoms when compared to the control cohort and lower dose cohort are indicative of the efficacy of the high dose treatment regimen in adult subjects.

5. Example 5 - Variable duration treatment regimens

This example describes the efficacy of administering single doses of allergens for varying treatment durations.

Experimental protocol

21 day old mice are fed milk (supplemented with high quantities of milk allergens as indicated) daily for 1, 2, 3, 4, 5 or 6 months. Control animals are fed either milk or PBS. After the treatment period, the mice are then administered a dose of allergen that is sufficient to elicit an allergic response. Tolerance to milk allergens is measured at the end of the experiment. Tolerance is determined by lack of specific IgE production, MMP-1 production and specific activation of T cells against milk allergens, and severity of clinical symptoms.

Groups of mice will be fed according to regimens of differing duration. However, the total amount of allergens administered each day will remain constant.

Sample Treatment Treatment duration (months)

Control 1 Milk 1

Control 2 Milk 2

Control 3 Milk 3

Control 4 Milk 4

Control 5 Milk 5 Control 6 Milk 6

Sample #1 Milk + 20-fold increased alpha_si -casein 1

Sample #2 Milk + 20-fold increased alpha_si -casein 2

Sample #3 Milk + 20-fold increased alpha_si -casein 3

Sample #4 Milk + 20-fold increased alpha_si -casein 4

Sample #5 Milk + 20-fold increased alpha_si -casein 5

Sample #6 Milk + 20-fold increased alpha_si -casein 6

Sensitisation and immunological assay

Results

Results showing no reaction or a less severe reaction amongst individuals of trial cohorts undergoing longer duration treatments when administered an amount of allergen sufficient to elicit an allergic response in the control group are indicative that the individual(s) of the trial cohort(s) have been tolerised, and thus of the efficacy of the longer duration treatment regimen. For example, results including an immunological profile amongst a 4, 5, or 6 month duration trial cohort indicative of a lack of specific IgE production, reduced MMP-1 production, and no specific activation of T cells against milk allergens, are indicative of tolerisation of the long duration trial cohorts.

Where in the foregoing description reference has been made to elements or integers having known equivalents, then such equivalents are included as if they were individually set forth.

Although the invention has been described by way of example and with reference to particular embodiments, it is to be understood that modifications and/ or improvements may be made without departing from the scope or spirit of the invention.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

INDUSTRIAL APPLICATION

The present invention provides methods and compositions for promoting a beneficial immune response in mammalian subjects, and is thus anticipated to provide significant medical and economic benefits, such as benefits to human health.

Claims

A method of decreasing a subject's risk of developing sensitivity to one or more bovine milk allergens, the method comprising orally administering to the subject one or more bovine milk allergens, wherein the administration is of a composition comprising one or more bovine milk allergens wherein the one or more bovine milk allergens is present at a concentration at least 50% greater than the concentration(s) of the milk allergen(s) in whole bovine milk; and optionally wherein the administration is one or more of the following:

The method according to claim 1 wherein the one or more bovine milk allergens is one or more components of bovine milk capable of increasing IgE production or levels in a subject exposed to same.

The method according to claim 1 or claim 2 wherein the one or more bovine milk allergens is one or more milk proteins, one or more milk protein fragments, including one or more allergenic peptides derived from a bovine milk protein.

The method according to any one of claims 1 to 3 wherein the one or more bovine milk proteins, milk protein fragments, or allergenic peptides is selected or derived from alphaSl- casein, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, or lactoferrin.

A method of modulating in a subject in need thereof an immune response to one or more bovine milk allergens, the method comprising administering to a subject not previously sensitised to one or more bovine milk allergens a composition comprising one or more bovine milk allergens wherein the one or more bovine milk allergens is present at a concentration at least 50% greater than the concentration(s) of the milk allergen(s) in whole bovine milk.

The method according to claim 5 wherein the subject not previously sensitised to one or more bovine milk allergens is a subject not previously directly exposed to one or more bovine milk allergens.

The method of any one of claims 1 to 6 wherein the administration is daily, twice-daily, three times daily, four times daily, or more than four times daily.

8. The method of any one of claims 1 to 7 wherein the administration is for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, or at least about six months.

9. The method of any one of claims 1 to 8 wherein the administration is

b) at least daily,

c) for at least one month.

10. The method of any one of claims 1 to 9 wherein sensitivity to one or more bovine milk

allergens is one or more of the following:

d) presence of one or more clinical symptoms of milk allergy.

11. The method of any one of claims 1 to 10 wherein the one or more milk proteins are

administered in the form of a composition with a physiologically acceptable diluent, adjuvant, carrier or excipient.

12. The method of claim 11 wherein in one embodiment, said physiologically acceptable diluent, adjuvant, carrier or excipient is a food.

13. The method of claim 12 wherein the food is cultured milk, yoghurt, cheese, milk drink or milk powder.

14. The method of any one of claims 1 to 11 wherein the composition is a pharmaceutical

composition and said excipient or diluent is pharmaceutically acceptable diluent, adjuvant, carrier or excipient.

15. The method of any one of claims 1 to 14 wherein the subject is a foetal, a neonatal, an infant, a child, a juvenile, or an adult subject.

16. The method of claim 15 wherein the subject is a foetal subject, the method comprises

administering the one or more milk proteins or a composition comprising one or more milk allergens to the foetal subject's mother.

17. The method of claim 15 wherein the subject is a breastfeeding neonatal, infant, or child

subject, the method comprises administering the one or more milk allergens or a composition comprising one or more milk allergens to the subject's mother.

8. The method of any one of claims 1 to 4 or 7 to 17 wherein the sensitivity is or exacerbates one or more of the following conditions: food protein-induced enterocolitis syndrome (FPIES), atoptic dermatitis, eosinophilic esophagitis, colic including infantile colic, isolated failure to thrive, lethargy, cutaneous reactions, including urticaria, angioedema, and pruritus, gastrointestinal symptoms including vomiting and diarrhoea, and respiratory symptoms including chronic rhinitis and recurrent wheezing.

19. The method of any one of claims 1 to 4 or 7 to 17 wherein the sensitivity is anaphylaxis.

20. The method of any one of claims 1 to 4 or 7 to 17 wherein the sensitivity or adverse immune response is or exacerbates one or more conditions associated with allergy, such as eczema including atopic eczema (also known as infantile eczema, flexural eczema or atopic dermatitis), xerotic eczema (also known as asteatotic eczema), seborrhoeric dermatitis, dyshidrosis, discoid eczema, venous eczema, Duhring's disease, or neurodermatitis.

21. The method of claim 5 or claim 6 wherein the modulation is downregulation of an existing immune response.

22. A composition comprising one or more bovine milk allergens at a concentration at least 50% greater, at least 75% greater, at least 100% greater, at least 200% greater, at least 300% greater, at least 400% greater, at least 500% greater, at least 600% greater, at least 700% greater, at least 800% greater, at least 900% greater, or at least 1000% greater than the concentration (s) of the milk allergen(s) in whole bovine milk.

23. The composition of claim 22 in which one of the one or more bovine milk allergens is

alphaSl-casein, the composition comprises alphaSl -casein at a concentration of at least about 15g/L, at least about 20g/L, at least about 30g/L, at least about 40g/L, at least about 50g/L, at least about 60g/L, at least about 70g/L, at least about 80g/L, at least about 90g/L, at least about lOOg/L.

24. The composition of claim 22 or 23 in which one of the one or more bovine milk allergens is alpha-lactoalbumin, the composition comprises alpha-lactalbumin at a concentration of at least about 1.5g/T, at least about 2g/L, at least about 3g/L, at least about 4g/T, at least about 5g/L, at least about 6g/L, at least about 7g/L, at least about 8g/L, at least about 9g/L, or at least about lOg/L.

25. The composition of any one of claims 22 to 24 in which one of the one or more bovine milk allergens is beta-lactoglobulin, the composition comprises beta-lactoglobulin at a

concentration of at least about 4.5g/L, at least about 6g/L, at least about 9g/L, at least about 12g/L, at least about 15g/L, at least about 18g/L, at least about 21g/L, at least about 24g/L, at least about 27g/L, or at least about 30g/L.

26. The composition of any one of claims 22 to 25 in which one of the one or more bovine milk allergens is bovine serum albumin, the composition comprises bovine serum albumin at a concentration of at least about 0.6g/L, at least about 0.8g/L, at least about 1.2g/L, at least about 1.6g/L, at least about 2g/L, at least about 2.4g/L, at least about 2.8g/L, at least about 3.2g/L, at least about 3.6g/L, or at least about 4g/L.

27. The composition any of one of claims 22 to 26 wherein the one or more bovine milk proteins comprise at least about 10% of the subject's recommended protein daily intake.

28. The composition of any one of claims 22 to 27 wherein the one or more bovine milk allergens is present in an amount sufficient to elicit tolerance.

29. The composition of any one of claims 22 to 28 wherein the amount sufficient to elicit

tolerance is an amount above the concentration range associated with risk of eliciting sensitivity to one or more milk allergens.

30. The composition of any one of claims 22 to 29 wherein the amount sufficient to elicit

tolerance is insufficient to elicit sensitivity.

31. The composition of claim 30 wherein the amount insufficient to elicit sensitivity is one or more of the following:

assessed by a determination of the presence of bovine milk allergen-specific IgE; c) an amount insufficient to elicit more than 0.35 kilounits of IgE/L as assessed by Pharmacia Diagnositcs UNICAP®, for example by Pharmacia Diagnostics

UNICAPIOO®;

d) an amount insufficient to elicit one or more clinical symptoms of milk allergy.

32. The composition of any one of claims 28 to 31 wherein the amount sufficient to elicit

tolerance is from about 2g to about 15g per day total milk protein allergen.

33. The composition according to any one of claims 22 to 32 wherein the composition is an infant formula, follow-on formula, growing-up formula or dietetic product, including hypo allergenic embodiments of such compositions.

34. The composition of any one of claims 22 to 33 comprising, consisting essentially of, or

consisting of fresh whole milk, fresh skim milk, recombined or reconstituted whole or skim milk powder, skim milk concentrate, skim milk retentate, concentrated milk, ultrafiltered milk retentate, milk protein concentrate (MPC), milk protein isolate (MPI), calcium depleted milk protein concentrate (MPC), casein, caseinate, colostrum, a colostrum extract, colostrum protein concentrate (CPC), colostrum whey, an immunoglobulin extract from colostrum, whey (including sweet whey, lactic acid whey, mineral acid whey, or reconstituted whey powder), whey protein isolate (WPI), whey protein concentrate (WPC), a composition derived from any milk or colostrum processing stream, a composition derived from the retentate or permeate obtained by ultrafiltration or microfiltration of any milk or colostrum processing stream, a composition derived from the breakthrough or adsorbed extract obtained by chromatographic (including but not limited to ion and gel permeation chromatography) separation of any milk or colostrum processing stream, extracts of any of these milk derivatives including extracts prepared by multistage fractionation, solvent fractionation, supercritical fractionation, near critical fractionation, distillation, centrifugal fractionation, hydrolysates of any of these materials, extracts of the hydrolysates, and any combination of any two or more of these materials, including combinations of hydrolysed and/ or non- hydrolysed extracts.

35. The composition of any one of claims 22 to 34 is a dairy product, for example a milk drink, comprising an added source of milk protein or milk allergen.

36. One or more milk allergens for treating or preventing sensitivity to one or more milk

allergens.

37. Use of one or more milk allergens in the manufacture of a composition for treating or

preventing sensitivity to one or more milk allergens.

38. The composition of any one of claims 22 to 35 or the use of claim 37 wherein the

composition is a unit dosage form.

39. The composition of any one of claims 22 to 35 or the use of claim 37 or 38 wherein the

composition is suitable for oral administration.

40. The use of claim 37 or 38 wherein the composition is suitable for parenteral administration.

41. The use of claim 37 wherein in embodiments relating to preventing eczema in a foetal subject, the composition is suitable for oral administration to a pregnant mother during gestation.